Diagnosis of prostate cancer

ABSTRACT

The invention provides methods for isolating RNA from whole urine and urine fractions for the diagnosis of prostate cancer and/or benign prostate hyperplasia. An exemplary method for diagnosing prostate cancer in an individual, said method comprises: (a) determining the amount of RNA encoding one or more diagnostic genes in the soluble urine fraction of a urine sample obtained from said individual; (b) comparing the amount of said RNA to a reference value for said one or more diagnostic genes, wherein said reference value is derived from the amount of RNA encoding said one or more diagnostic genes in one or more individuals that do not have prostate cancer; and (c) diagnosing said individual as having prostate cancer when the amount of said RNA is greater than said reference value.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/997,868, filed Sep. 9, 2013, now U.S. Pat. No. 9,663,781, which is aU.S. national stage of PCT/US2011/067880, filed Dec. 29, 2011, whichclaims priority to U.S. Provisional Application No. 61/428,750, filed onDec. 30, 2010, incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

This invention relates to methods for isolating, processing, andidentifying nucleic acids from biological samples, including urine, fordiagnosing prostate disease including prostate cancer.

BACKGROUND OF THE INVENTION

Prostate cancer is a cancer of the prostate gland, a small, walnut-sizedstructure making up a part of a man's reproductive system that wrapsaround the urethra. Early detection of prostate cancer, for example,prior to presentation of symptoms, can improve patient prognosis. It isestimated that there are 200,000 new cases and 25,000 deaths fromprostate cancer each year in the United States. Jemal A, et al. CACancer J Clin. 2008; 58(2):71-96. Cancer cells with a Gleason grade 3 or4 generally indicate aggressive cancers that require treatment. Theprostate-specific antigen (PSA) test measures the amount of PSA in ablood sample of a patient and is used to determine if a patient shouldreceive a biopsy test. However, the PSA test detects both benignprostate hyperplasia (BPH) and prostate cancer, therefore only 20-30% ofbiopsies are found to be positive for cancer. Out of those patients withnegative results from a first biopsy, approximately 10% of secondbiopsies are found to be positive for cancer. Guyon, I. et al. UrotodayInt J. 2009 August; 2(4). In addition, biopsy is an invasive procedurethat can result in complications such as septicemia, infections,hemorrhages, hematomas, arteriovenous fistula, tumor dissemination,bladder perforation, urinary obstruction, severe pain, strokes, perineumabscesses, erectile dysfunction, loss of desire and reduced sexualactivity.

SUMMARY OF THE INVENTION

The present invention relates to methods for diagnosing prostate cancer(PCa) and/or distinguishing prostate cancer from benign prostatehyperplasia (BPH) in an individual by assessing urinary RNA levels.

In one aspect, the invention provides a method for diagnosing prostatecancer in an individual, the method comprises: (a) determining theamount of RNA encoding one or more diagnostic genes in the soluble urinefraction of a urine sample obtained from said individual; (b) comparingthe amount of said RNA to a reference value for the one or morediagnostic genes, wherein the reference value is derived from the amountof RNA encoding the one or more diagnostic genes in one or moreindividuals that do not have prostate cancer; and (c) diagnosing theindividual as having prostate cancer when the amount of said RNA isgreater than the reference value.

The RNA in the soluble urine fraction may be concentrated to form asoluble urine concentrate by any suitable means including, for example,ultrafiltration (e.g., using a filter membrane having a cutoff of about100 kDa, 50 kDa, 10 kDa, or 3 kDa), lyophilization, dialysis, or othermeans for dehydration or concentration. In some embodiments, the solubleurine fraction is obtained by concentrating whole urine obtained fromthe individual by ultrafiltration. In some embodiments, the solubleurine concentrate is formed by ultrafiltration of the soluble urinefraction through a membrane filter having a suitable molecular weightcutoff; such as a filter having a nominal molecular weight limit of notmore than about 50,000 daltons; such as a filter having a nominalmolecular weight limit of not more than about 3,000 daltons. In stillother embodiments, the soluble urine fraction produced by theultrafiltration is cell-free.

Ultrafiltration may be performed using a syringe filter or a centrifugalfiltration unit. In some embodiments, the membrane filter has a netpositive or net neutral charge. Suitable membrane materials include, forexample, cellulose based materials (e.g. regenerated cellulose,methylcellulose, cellulose triacetate), polysulfone, andpolyethersulfone. Dialysis may be performed against any suitablecounter-solvent including for, example, polyethylene glycol, and thedialysis membranes are designed to have any appropriate molecular weightcutoff, as described herein. In embodiments which use lyophilization toform the soluble urine concentrate, the urine sediment is physicallyseparated from the soluble urine fraction prior to lyophilization.Optionally, the lyophilized product containing the RNA from the solublefraction is resuspended (solubilized) following lyophilization in avolume of diluent less than the original volume of the urine sample.

The RNA from the soluble urine concentrate may be isolated by anysuitable method including, for example, solid phase extraction.Optionally, the RNA is subsequently released from the solid phase forfurther processing.

In one example, the above methods further include detecting the RNA(e.g., mRNA) from the soluble urine concentrate. Exemplary methods fordetecting RNA include reverse transcription coupled with real-time PCR,northern blot, UV spectroscopy, hybridization of RNA or cDNA to a probesuch as in microarray or flow cytometry.

Urine may be obtained from any individual. An individual may be healthyand without any known disease. Alternatively, an individual may be aperson suspected of having a disease. Urine samples may be pooled frommultiple individuals or from multiple samples obtained from a singleindividual. In the latter case, the combined sample may represent thetotal daily urinary output of a single individual. Preferably, urinesamples are collected in sterile containers in order to minimize thepossibility for contamination by environmental microorganisms or otherforeign matter. In one embodiment, the urine sample is obtained using acatheter.

In some embodiments, at least one of the diagnostic genes is heat shock60 kDa protein 1 (HSPD1), inosine monophosphate dehydrogenase 2(IMPDH2), PDZ and LIM domain 5 (PDLIM5), UDP-N-acteylglucosaminepyrophosphorylase 1 (UAP1), and prostate cancer antigen-3(PCA3). In someembodiments the diagnostic genes comprise each of HSPD1, IMPDH2, PDLIM5,and UAP1.

The amount of RNA encoding one or more diagnostic genes present in thesoluble urine fraction is determined by reverse-transcriptase PCR(RT-PCR). For example, the determination by RT-PCR can be performed inreal-time. In some embodiments, the Ct value is used to determine theamount of one or more of said diagnostic gene RNA.

Optionally, the amount of one or more of the diagnostic gene RNA isnormalized to the amount of control gene RNA. The control gene RNA maybe, for example, RNA encoding prostate-specific antigen (PSA), c-abloncogene 1, receptor tyrosine kinase (ABL1), beta actin (ACTB), beta-2microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH),and beta glucuronidase (GUSB), or fragments thereof.

In some embodiments, the amount of one or more of said diagnostic genesis normalized to the amount of control gene RNA using the formula:Normalized Diagnostic Gene Score=Ln(Diagnostic Gene/Control Gene). Insome embodiments, the amount of two or more diagnostic gene RNAs aredetermined. In certain embodiments, the amount of the diagnostic geneRNAs are used to generate a single diagnostic score, wherein the singlediagnostic score is compared against a reference value for the singlediagnostic score in order to diagnose said individual. In certainembodiments, the amount of each of the diagnostic gene RNAs isnormalized to the amount of control gene RNA using the formula:Normalized Diagnostic Gene Score=Ln(Diagnostic Gene RNA/Control GeneRNA). In some embodiments, the single diagnostic score is calculated asthe sum of the individual Normalized Diagnostic Gene Scores.

In some embodiments the control gene RNA encodes PSA or GAPDH. In someembodiments, the reference value is between about 2.6 and about 5.0;such as about 2.6 or about 5.0.

Optionally, an individual diagnosed as having prostate cancer isindicated for prostate cancer therapy. In certain embodiments, prostatecancer therapy is initiated on the individual following a positivediagnosis.

As used herein the term “diagnosing,” “diagnosis,” and the like meansdetermining a disease state or condition in an individual in such a wayas to inform a health care provider as to the necessity or suitabilityof a treatment for that individual. Optionally, an individual for whicha specific diagnosis is made is further indicated for treatment for thatdisease state or condition. Optionally, treatment of the individual isinitiated based on that diagnosis.

As used herein the term “soluble fraction of urine” means urine which issubstantially free (preferably, less than about 1% w/w) of cells,cellular debris, organelles, organisms, and insoluble matter (e.g.,mineral crystals). Typically, an unprocessed urine sample obtained froman individual is a mixture of the soluble fraction and the urinesediment, with the soluble fraction making up the largest portion of themixture. Under normal conditions, the material that makes up the urinesediment is suspended in the soluble fraction and requires processing toeffect useful separation. Preferably, the soluble fraction of urine andthe resulting soluble urine concentrate are acellular (i.e., lackingcells). It is understood that the urine fraction may be renderedacellular (e.g., by filtration and/or centrifugation) withoutnecessarily removing all other insoluble matter such as organelles,cellular debris, and insoluble matter.

As used herein the term “urine sediment” means that fraction of urinecomprising cells, cellular debris, organelles, organisms, and/orinsoluble matter that may be removed from the soluble fraction of urine.Exemplary methods for separating urine sediment from soluble fraction ofurine include centrifugation, filtration, and/or sedimentation undergravity.

As used herein the term “soluble urine concentrate” means that solublefraction of urine which is substantially free (preferably, less thanabout 1% w/w) of cells, cellular debris, organelles, and organisms andwhere the volume of the soluble fraction of urine has been reduced by atleast 50%, at least 60%, at least 75%, at least 80%, at least 90% ormore from the original urine volume.

As used herein the term “ultrafiltration” means a separation processwhich includes a filtration through a semi permeable membrane under apositive pressure such that solutes of higher molecular weight areretained by the membrane while water and low molecular weight solutespass though the membrane. Exemplary positive pressure includes but notlimited to hydrostatic pressure, centrifugal force.

As used herein the term “nominal molecular weight limit” in the contextof a filter membrane means a pore size where over 90% of the solute withthat molecular weight will be retained. Exemplary nominal molecularweight limit suitable for concentrating soluble urine fractioncomprising RNA include 3 kDa, 10 kDa, 30 kDa, 50 kDa and 100 kDa.

The term “RNA” is meant to include mRNA, tRNA, and rRNA. In preferredembodiments, the RNA is mammalian RNA (e.g., RNA obtained from mammalianurine). In other embodiments, the RNA is non-viral.

“Primer” refers to an oligonucleotide that hybridizes to a substantiallycomplementary target sequence and is capable of acting as a point ofinitiation of DNA synthesis when placed under conditions in which primerextension is initiated (e.g., primer extension associated with anapplication such as PCR). An oligonucleotide “primer” may occurnaturally, as in a purified restriction digest or may be producedsynthetically. Primers are typically between about 10 and about 100nucleotides in length, preferably between about 15 and about 60nucleotides in length, more preferably between about 20 and about 50nucleotides in length, and most preferably between about 25 and about 40nucleotides in length. An optimal length for a particular primerapplication may be readily determined in the manner described in H.Erlich, PCR Technology, Principles and Application for DNA Amplification(1989).

A “probe” refers to an oligonucleotide that interacts with a targetnucleic acid via hybridization. A probe may be fully complementary to atarget nucleic acid sequence or partially complementary. The level ofcomplementarity will depend on many factors based, in general, on thefunction of the probe. A probe or probes can be used, for example todetect the presence or absence of an RNA or cDNA in a sample by virtueof the sequence characteristics of the target. Probes can be labeled orunlabeled, or modified in any of a number of ways well known in the art.A probe may specifically hybridize to a target nucleic acid. Probes canbe designed which are between about 10 and about 100 nucleotides inlength and hybridize to the target nucleic acid such as RNA or cDNA.Oligonucleotides probes are preferably 12 to 70 nucleotides; morepreferably 15-60 nucleotides in length; and most preferably 15-25nucleotides in length. The probe may be labeled with a detectable label.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic of the experimental design of isolating RNAfrom urine sediment, urine filtrate (after removing cells from urine byfiltration) and whole urine. Three different RNA isolation proceduresare illustrated.

FIG. 2 is a bar graph showing the relative amount of PSA RNA recoveredfrom various urine fractions and the diagnostic score calculated fromthe GADPH-normalized levels of four genes associated with prostatecancer. Experimental details are provided in Example 7.

FIG. 3 is a series of graphs showing the 4-gene score of HSPD1, UAP2,IMPDH2, and PDLIM5, normalized to GAPDH, in urine supernatant (top) andwhole urine (bottom) samples obtained from male patients diagnosed ashaving BPH or PCa. Each bar graph indicates the proportion ofindividuals, for each condition, having 4-gene scores above diagnosticcut-off threshold values of 2.6 and 5.0.

FIG. 4 is a bar graph showing the relative amount of PSA (multiplied bya factor of ten and normalized to GAPDH) and PCA3 measured in wholeurine, urine sediment, and urine supernatant obtained from male patientsdiagnosed as have PCa. Error bars represent S.E.M.

FIG. 5 is a bar graph showing the median difference in the 4-gene scorenormalized to PSA RNA levels in the three urine fractions betweensamples obtained from BPH and PCa patients.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for diagnosing prostate cancer(PCa) and/or distinguishing prostate cancer from benign prostatehyperplasia (BPH) in an individual. The method assesses the RNA from onemore diagnostic genes in the soluble urine fraction; optionally asoluble urine concentrate. In some embodiments, the amount of diagnosticgene RNA is used to generate a diagnostic score which is compared to areference value for that diagnostic score in order to inform thediagnosis.

Diagnostic Genes

The RNA from any gene having a known association (over-expression orunder-expression) with prostate cancer may be assessed in the methods ofthe present invention. Without wishing to be bound by any theory, it isbelieved that malignant cells die and release their contents into thesoluble urine fraction and/or are shed into the urine where the cellsdeteriorate, releasing their contents, including nucleic acids. Theamount of diagnostic gene RNA may be increased in the soluble urinefraction by palpating the prostate (i.e., by a digital rectalexamination) prior to urine sample collection. Some diagnostic genessuitable for analysis in the present diagnostic methods are providedbelow. Additional diagnostic genes are disclosed in U.S. PatentPublication 2009/0226915 and Guyon et al. (Urotoday Int. J. 2(4), 2009).

Heat Shock 60 kDa Protein-1 (HSPD1):

HSPD1 is a mitochondrial molecular chaperone which mediates proteinfolding in normal cells and may function as a signaling molecule in theinnate immune system. In one example, the cDNA encoding human HSPD1 isprovided at Genbank Accession No. NM₁₃ 002156.

Inosine Monophosphate Dehydrogenase-2 (IMPDH2):

IMPDH2 is the rate-limiting enzyme in the de novo guanine nucleotidebiosynthesis. The enzyme is involved in maintaining cellular guaninedeoxy- and ribonucleotide pools needed for DNA and RNA synthesis bycatalyzing the NAD-dependent oxidation of inosine-5′-monophosphate intoxanthine-5′-monophosphate, which is then converted intoguanosine-5′-monophosphate. In one example, the cDNA encoding humanIMPDH2 is provided at Genbank Accession No. NM_000884.

PDZ and LIM Domain-5 (PDLIM5):

PDLIM5 encodes a LIM domain protein (i.e., cysteine-rich double zincfingers) that act as scaffolds for the formation of multiproteincomplexes for cytoskeleton organization, cell lineage specification, andorgan development. The encoded protein is also a member of the Enigmaclass of proteins, a family of proteins that possess a 100-amino acidPDZ domain in the N terminus and 1 to 3 LIM domains in the C terminus.Several PDLIM5 variants are known. The cDNA encoding some human PDLIM5variants are provided at Genbank Accession No. NM_006457, NM_001011516,NM_001011513, and NM_001011515.

UDP-N-acetylglucosamine pyrophosphorylase-1 (UAP1):

UAP1 is also known as AgX-1 and encodes an enzyme involved inaminosugars metabolism. In one example, the cDNA encoding human UAP1 isprovided at Genbank Accession No. S73498.

Control Genes

The absolute amount of RNA from any one or more diagnostic genes presentin urine (or a urine fraction) may be informative of a diagnosis.However, it may be desirable to control for inter- and intra-individualdifferences in the amount of urinary RNA. Thus, the amount of diagnosticgene RNA may be normalized either to the total amount of RNA present inthe urine sample (or urine fraction), or to the amount of RNA from agene that is not associated with the disease state (e.g., prostatecancer). Suitable control genes may be identified by comparing the RNAlevels in the urine fraction of interest from normal individuals andindividuals diagnosed as having disease. Those candidate genes that havesubstantially the same levels between the two groups may be useful ascontrol genes. Suitable control genes include, for example, c-abloncogene 1 (c-abl1), receptor tyrosine kinase (ABL1), beta actin (ACTB),beta-2 microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase(GAPDH), and beta glucuronidase (GUSB).

Prostate-specific antigen (PSA) may be used either as a control gene oras a diagnostic gene. PSA is well-known to be over-expressed andreleased from prostate tumors and is commonly assessed in blood, whereinelevated levels indicate prostate cancer. Thus, the amount of PSA RNA inurine also may be taken as an indicator of prostate cancer.Alternatively, the ratio of one or more diagnostic genes to PSA in urineis also informative on the diagnosis of prostate cancer. Thus, PSA mayserve as the control/comparator gene in these cases.

Urine Samples

A urine sample typically consists of a soluble fraction and a sedimentfraction. The sediment fraction may contain cells, cellular debris,organelles, microorganisms, and/or insoluble minerals (e.g., kidneystones). Soluble urine fraction is substantially free (less than 1% w/w)of urine sediment and preferably contains only soluble molecules (e.g.,urea, nucleic acids, soluble proteins, etc.). Urine samples may beobtained from healthy individuals (i.e., free of known disease) orindividuals known or suspected to have a disease or other condition.Alternatively, a urine sample may consist of urine samples pooled fromseveral individuals.

Methods for Separating Urine Sediment from Soluble Urine Fraction

The urine sediment may be separated from the soluble urine fraction byany convenient method including, for example, centrifugation,sedimentation under gravity, or filtration. In one example,centrifugation can be performed at 1000×g to 30,000×g for 10 minutes topellet the urine sediment and some or all of the soluble urine fractionmay be removed. In another example, urine sediments can be separated byfiltration using relatively high molecular weight cutoff filters suchthat the urine sediment is retained on the filter membrane while thesoluble urine fraction including the soluble RNA passes into thefiltrate. Exemplary filter membranes can be made of cellulose basedmembranes (e.g. regenerated cellulose, methylcellulose, cellulosetriacetate), polysulfone, polyethersulfone. Commercial kits such as ZRUrine RNA Isolation Kit™ (ZYMO Research Corporation) are available toremove urine sediments from soluble urine fraction.

Methods for Concentrating Soluble Urine Fractions

Soluble urine fractions may be concentrated by any convenient methodsuitable for the volume of urine to be processed and the anticipatedsize of the soluble RNA to be identified and isolated. Suitableconcentration methods include, for example, ultrafiltration,lyophilization, and dialysis (e.g., against polyethylene glycol).Ultrafiltration, involves filtration though a semi permeable membraneunder a positive pressure such as hydrostatic pressure or centrifugalforce such that solutes of higher molecular weight remain in theretentate while water and low molecular weight solutes pass into thefiltrate. Typically, the membranes used for concentration have a smallerpore diameter (e.g., lower molecular weight cutoff) than the filtersused to remove the urine sediment.

Preferably, the semipermeable membrane materials used for concentratingsoluble urine fractions do not bind or retain soluble RNA. Suitablematerials include, for example, cellulose based materials (e.g.regenerated cellulose, methylcellulose, cellulose triacetate),polysulfone, and polyethersulfone. The semipermeable membranes areavailable in various pore sizes. The pore size where over 90% of thesolute with that molecular weight will be retained is termed as “nominalmolecular weight limit” (NMWL). Exemplary nominal molecular weightlimits suitable for isolating RNA from urine include 3 kDa, 10 kDa, 30kDa, 50 kDa and 100 kDa. The pore size of semipermeable membranesinclude nominal molecular weight limits that can range from about 1 kDato about 200 kDa, from about 2 kDa to about 150 kDa, and from about 3kDa to about 100 kDa. Table 1 below provides general guidance forselecting the membrane for retention of RNA based on the nucleotidecontent of a nucleic acid. Alternatively, the soluble RNA is retained onthe filter and later recovered. Suitable membranes for soluble RNAretention include anionic membranes such as PVDF.

TABLE 1 NMWL guidelines for selecting semipermeable membrane forultrafiltration. Single-stranded nucleotide Double-stranded nucleotideNMWL cut-off (bases) cut-off (base pair)  3 kDa 10 10 10 kDa 30 20 30kDa 60 50 50 kDa 125 100 100 kDa  300 125

Various commercially available ultrafiltration kits and devices areavailable to concentrating a sample such as Amicon Ultra-4 CentrifugalFilter Units, Amicon Ultra-15 Centrifugal Filter Units, Centricon®centrifugal filter devices (Millipore, Mass., USA), Pierce Concentrator(Thermo Fisher Scientific, Ill., USA). In one example, 15 ml of solubleurine fraction can be concentrated to 500 μl using Amicon Ultra-15Centrifugal Filter Units by centrifugation for 30 minutes at 4000×g.

In one example, soluble urine fraction can be concentrated bylyophilization. Lyophilization is a freeze-drying process that works byfreezing the material and then reducing the surrounding pressure andadding enough heat to allow the frozen water in the material to sublimedirectly from the solid phase to gas. Lyophilization machines areavailable from commercial vendors such as Labconco (MO, USA), MillrockTechnology (NY, USA).

In another example, soluble urine fraction can be concentrated byplacing the soluble urine fraction by dialysis against a solutioncontaining polyethylene glycol and using a dialysis bag with appropriatemolecular cutoff. The molecular weight cutoff can range from 3 kDa-100kDa depending on the size of RNA to be retained within the dialysis bag.Appropriate dialysis tubings can be obtained commercially such asSigma-Aldrich, Thermo-Scientific.

Methods for RNA Isolation and Extraction

RNA may be isolated and extracted from aqueous samples such as solubleurine fraction or soluble urine concentrate using standard techniques,see, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual,Second Edition (1989), Cold Spring Harbor Press, Plainview, N.Y.Particularly useful are solid phase extraction methods. Reagents andkits for isolating RNA from a biological sample are commerciallyavailable e.g., RNeasy Maxi Kit, RNeasy Protect Mini kit, RNeasy ProtectCell Mini kit, QIAamp RNA Blood Mini kit, from Qiagen; MELT™,RNaqueous®, ToTALLY RNA™, RiboPure™-Blood, Poly(A)Purist™ from AppliedBiosystems; TRIZOL® reagent, Dynabeads® mRNA direct kit from Invitrogen.In one example, kits provided by Qiagen employ silica resin to bindnucleic acid including RNA. RNA in the solution binds to the silicaresin while the proteins and other solutes passes through. After severalsteps of washing, RNA can be eluted using the buffer provided by themanufacturer. In one example, NucliSENS® easyMAG® automated system(bioMérieux, Inc., NC, USA) may be used for the extraction of totalnucleic acids including RNA. RNA from soluble urine fraction or solubleurine concentrate will bind to NucliSENS® magnetic silica particles. TheRNA bound to the magnetic silica particles will be washed with washbuffer supplied by the manufacturer and will be eluted from the magneticsilica particles by heating using manufacturer's protocol. In anotherexample, RNA can be isolated by adsorbing on an anion exchange resinfollowed by elution with high salt buffer. Exemplary anion exchangeresins include Diethylaminoethyl (DEAE) crosslinked to polystyrene orcellulose, DNAPac® series of polymer-based anion-exchange columns fromDionex, anion exchange columns from Thermo Scientific.

Reverse Transcription of RNA to cDNA

Various methods to reverse transcribe RNA to cDNA are known in the art.Various reverse transcriptases may be used, including, but not limitedto, MMLV RT, RNase H mutants of MMLV RT such as Superscript andSuperscript II (Life Technologies, GIBCO BRL, Gaithersburg, Md.), AMVRT, and thermostable reverse transcriptase from Thermus Thermophilus. Inone example, RNA extracted from soluble urine fraction or soluble urineconcentrate may be reverse transcribed to cDNA using the protocoladapted from the Superscript II Preamplification system (LifeTechnologies, GIBCO BRL, Gaithersburg, Md., catalog no: 18089-011), asdescribed by Rashtchian, A., PCR Methods Applic. (1994), 4:S83-S91. Themethod is described below.

One (1) to five (5) micrograms of RNA extracted from soluble urinefraction or soluble urine concentrate in 13 μl of DEPC-treated water isadded to a clean microcentrifuge tube. One microliter of either oligo(dT) (0.5 mg/ml) or random hexamer solution (50 ng/μl) is added andmixed gently. The mixture is then heated to 70 degrees centigrade for 10minutes and then incubated on ice for one minute. Then, it iscentrifuged briefly followed by the addition of 2 μl of 10× Synthesisbuffer (200 mM Tris-HCl, pH 8.4, 500 mM KCl, 25 mM magnesium chloride, 1mg/ml of BSA), 1 μl of 10 mM each of dNTP mix, 2 μl of 0.1 M DTT, 1 μlof SuperScript IT RT (200 U/μl) (Life Technologies, GIBCO BRL,Gaithersburg, Md.). After gentle mixing, the reaction is collected bybrief centrifugation, and incubated at room temperature for 10 minutes.The tube is then transferred to a 42° C. water bath or heat block andincubated for 50 minutes. The reaction is then terminated by incubatingthe tube at 70° C. for 15 minutes, and then placing it on ice. Thereaction is collected by brief centrifugation, and 1 μl of RNase H (2units) is added followed by incubation at 37° C. for 20 minutes beforeproceeding to nucleic acid amplification.

In another example, reverse transcription of RNA to cDNA was combinedwith the RT-PCR reaction using RNA UltraSense® one-step real-time (RT)PCR System (Invitrogen).

Detection of RNA

The presence or amount of RNA isolated from soluble urine fraction orsoluble urine concentrate can be determined by several methods known inthe art. In one example, RNA can be detected by Northern blot. See,e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual, SecondEdition (1989), Cold Spring Harbor Press, Plainview, N.Y. In anotherexample, RNA can be detected by reverse transcription coupled with PCR,including real-time PCR. The cDNA is amplified in a real-time PCRreaction using gene specific primers. Real-time PCR detects the copynumber of PCR templates such as cDNA in a PCR reaction. Exemplarymethods for quantification of RNA by real-time PCR is described by Nolanet al. (Nat Protoc. 2006; 1(3):1559-82) and Gertsch et al. (Pharm Res.2002 August; 19(8):1236-43). The references are incorporated herein byreference. In another example, RT-PCR is performed in a combination witha reverse transcription of RNA to cDNA reaction using RNA UltraSense®one-step real-time (RT) PCR System (Invitrogen).

In one example, amplification of cDNA is monitored by SYBR green dye.The dye binds to double-stranded (ds)DNA in PCR, causing fluorescence ofthe dye. An increase in DNA product during PCR therefore leads to anincrease in fluorescence intensity and is measured at each cycle, thusallowing DNA concentrations to be quantified.

In another example, amplification of cDNA is monitored by TaqMan® probes(Heid et al., Genome Res. 1996; 6: 986-994). TaqMan® probes are based onthe principle of fluorescence quenching and involve a donor fluorophoreand a quenching moiety. The term “fluorophore” as used herein refers toa molecule that absorbs light at a particular wavelength (excitationfrequency) and subsequently emits light of a longer wavelength (emissionfrequency). The term “donor fluorophore” as used herein means afluorophore that, when in close proximity to a quencher moiety, donatesor transfers emission energy to the quencher. As a result of donatingenergy to the quencher moiety, the donor fluorophore will itself emitless light at a particular emission frequency than it would have in theabsence of a closely positioned quencher moiety.

The term “quencher moiety” as used herein means a molecule that, inclose proximity to a donor fluorophore, takes up emission energygenerated by the donor and either dissipates the energy as heat or emitslight of a longer wavelength than the emission wavelength of the donor.Suitable quenchers are selected based on the fluorescence spectrum ofthe particular fluorophore. Useful quenchers include, for example, theBlack Hole™ quenchers BHQ-1, BHQ-2, and BHQ-3 (Biosearch Technologies,Inc.), TAMRA, 4-(4-dimethylaminophenylazo) benzoic acid (DABCYL), andthe ATTO-series of quenchers (ATTO 540Q, ATTO 580Q, and ATTO 612Q;Atto-Tec GmbH). TaqMan® probes are designed to anneal to an internalregion of a PCR product. When the polymerase (e.g., reversetranscriptase) replicates a template on which a TaqMan® probe is bound,its 5′ exonuclease activity cleaves the probe. This ends the activity ofthe quencher (no FRET) and the donor fluorophore starts to emitfluorescence which increases in each cycle proportional to the rate ofprobe cleavage. Accumulation of PCR product is detected by monitoringthe increase in fluorescence of the reporter dye. If the quencher is anacceptor fluorophore, then accumulation of PCR product can be detectedby monitoring the decrease in fluorescence of the acceptor fluorophore.

To ensure accuracy in the quantification, it is usually necessary tonormalize expression of a target gene to one or more reference genesthat are stably expressed. Exemplary reference genes include beta actin(ACTB), beta-2 micro globulin (B2M), glyceraldehyde-3-phosphatedehydrogenase (GAPDH). Relative concentrations of DNA present during theexponential phase of the reaction are determined by plottingfluorescence against cycle number on a logarithmic scale (so anexponentially increasing quantity will give a straight line). Athreshold for detection of fluorescence above background is determined.The cycle at which the fluorescence from a sample crosses the thresholdis called the cycle threshold, Ct. A lower Ct value indicates highercopy number of an RNA. Amounts of RNA is determined by comparing theresults to a standard curve produced by real-time PCR of serialdilutions of a known amount of RNA or DNA.

Detection by Hybridization.

RNA isolated from soluble urine fraction or soluble urine concentratecan be detected following reverse transcription and amplification byhybridization with a nucleic probe that hybridizes specifically to theRNA of interest (i.e., a target RNA). The methods of the presentinvention can incorporate all known methods and means and variationsthereof for carrying out DNA hybridization, see, e.g., Sambrook, et al.,1989, Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor Press, Plainview, N.Y.

The RNA or cDNA may form a complex on a solid support prior to beingdetected. The complex may comprise a capture probe anchored to a solidsupport, the RNA of interest hybridized to the capture probe, and adetectably labeled probe hybridized to the RNA of interest. In somecases, the solid support may comprise a first member of a binding pairand the capture probe may comprise a second member of the binding pair.The binding of the first member of the binding pair to the second memberof the binding pair may anchor the capture probe to the solid support.Examples of solid support include but are not limited to beads,microparticles, microarray plates, microwells. Examples of binding pairinclude but are not limited to biotin/streptavidin, ligand-receptor,hormone-receptor, and antigen-antibody.

RNA and/or cDNA can be detected by performing an array-basedhybridization to detect the genes of interest in a sample, or todiagnose a disease in an individual. The resolution of array-basedmethod is primarily dependent upon the number, size and map positions ofthe nucleic acid elements within the array, which are capable ofhybridizing to the RNA. Microarrays are available commercially thatcover all human genes. For example, GeneChip® Human Exon 1.0 ST Arrayfrom Affymetrix (CA, USA), Whole Human Genome Microarray Kit fromAgilent Technologies (CA, USA) are capable of evaluating gene expressionof all known transcripts in human.

Alternatively, the hybridized complexes can also be detected using flowcytometry. Flow cytometry is a technique well-known in the art. Flowcytometers hydrodynamically focus a liquid suspension of particles(e.g., cells or synthetic microparticles or beads) into an essentiallysingle-file stream of particles such that each particle can be analyzedindividually. Flow cytometers are capable of measuring forward and sidelight scattering which correlates with the size of the particle. Thus,particles of differing sizes may be used in invention methodssimultaneously to detect distinct nucleic acid segments. In additionfluorescence at one or more wavelengths can be measured simultaneously.Consequently, particles can be sorted by size and the fluorescence ofone or more fluorescent labels probes can be analyzed for each particle.Exemplary flow cytometers include the Becton-Dickenson ImmunocytometrySystems FACSCAN. Equivalent flow cytometers can also be used in theinvention methods.

Example 1 Detection of RNA from Large Volume of Liquid Sample

The ability to detect RNA in a large volume of liquid sample was testedby adding 5 μl of RNA (845.6 ng/μl) to 15 ml of Tris-EDTA (TE) buffer.The resulting RNA solution was concentrated to less than 500 μl bycentrifugation using Amicon Ultra-15 filter units with nominal molecularweight limit of 3 kDa and 10 kDa (Millipore, Mass., USA). Theconcentration of RNA in the retentate from two different membranefilters were determined using NanoDrop™ spectrophotometer (ThermoScientific), which requires small volume of sample for analysis. Therecovery of RNA were comparable from the two membrane filter types: 94%for 10 kDa and 100% for 3 kDa. The results are shown in Table 3 below.

TABLE 2 Retention of spiked cell line RNA in TE measured by Nanodropconcentration. Volume Conc. Sample (μl) (ng/μl) Total (ng) % RecoverySpiked RNA/15 ml TE 5 845.6 4228 10 kDa membrane retentate 160 24.9 398494% 3 kDa membrane retentate 270 15.7 4239 100%

Example 2 Comparison of Recovery of Spiked RNA from Membranes withDifferent Pore Sizes

The range of filter pore sizes that can be used to concentrate the RNAwere evaluated for RNA retention using filter columns ranging from 3kDa-100 kDa. A known amount of cell line RNA (34 μg) was spiked into alarge volume of TE buffer (75 ml), split into five aliquots for astarting amount of 6.8 μg of total RNA per 15 ml aliquot. Each 15 mlaliquot was concentrated through five separate filter columns withdifferent pore sizes (nominal molecular weight limit: 3 kDa, 10 kDa, 30kDa, 50 kDa and 100 kDa retention, respectively). After concentrationwith the filter columns, the % recovery was determined. First, RNA yieldwas calculated by multiplying the final volume of sample by the finalconcentration of the sample measured by nanodrop. Second, the RNA yieldwas divided by the starting amount of RNA (6.8 μg) to give the final %recovery of each filter column. Based on these results, the 3 kDa poresize gave the highest recovery of 94%, followed by 10 kDa (87%), 30 kDa(78%), 50 kDa (80%), and 100 kDa with the lowest and final yield (67%).The results are shown in Table 3 below.

TABLE 3 Retention of spiked cell line RNA in TE measured by Nanodropconcentration. Volume Conc. % Sample (μl) (ng/μl) Total (ng) RecoverySpiked RNA/15 ml TE 5 1360 6800 100 kDa membrane retentate 135 33.7 455067% 50 kDa membrane retentate 206 26.4 5438 80% 30 kDa membraneretentate 290 18.4 5336 78% 10 kDa membrane retentate 190 31.3 5947 87%3 kDa membrane retentate 428 15.0 6420 94%

Example 3 Comparison of Recovery of Endogenous Urine RNA from Membraneswith Different Pore Sizes

Some factors may effect the efficiencies in retention of RNA in a realsample with endogenous RNA versus a sample spiked with RNA. Thesefactors include the presence of partially degraded or fragmented RNA andthe presence of urine RNases that may degrade RNA prior to processing.The ability of membranes with different pore sizes to retain endogenousurine RNA was evaluated. Whole urine (75 ml) was obtained from fiveseparate donors and split into five 15 ml aliquots per donor. Each ofthe five aliquots per donor was concentrated using the filters of fivedifferent pore sizes (nominal molecular weight limit: 3 kDa, 10 kDa, 30kDa, 50 kDa, and 100 kDa). RNA from each sample of concentrated urinewas extracted using EasyMag. Amplification of two different transcripts(GAPDH and ABL1) was performed for each sample by RT-PCR. In order toquantitate retention efficiencies, RNA concentrated from the topperforming filter column in the RNA spiking studies (3 kDa) was used asthe baseline (100%) for each donor. Using the cycle threshold (Ct)obtained by qRT-PCR, the recoveries for the 10 kDa-100 kDa filtercolumns were calculated based on the 3 kDa Ct values that were set at100%. Based on these results, retention of endogenous RNA was dependenton both the donor and the transcript, with all pore sizes above 3 kDademonstrating significantly reduced efficiency. The average retentionfor pore sizes 10 kDa-100 kDa for transcript 1 ranged from 32%-47% and16%-31% for transcript 2. The results are shown in Table 4 below.

TABLE 4 Recovery of endogenous urine RNA using filter column membraneswith different pore sizes. Transcript 1 Transcript 2 Donor 3 kDa 10 kDa30 kDa 50 kDa 100 kDa 3 kDa 10 kDa 30 kDa 50 kDa 100 kDa 1 100% 66% 59%79% 43% 100% 25% 39% 36%  9% 2 100% 67% 69% 79% 59% 100% 47% 40% 50% 19%3 100% 40% 43% 24% 25% 100% 28% 28%  9% 12% 4 100% 26% 20% 10%  4% 100%26% 25% 12%  8% 5 100% 38% 24% 34% 28% 100% 27% 11% 13% 31% Avg 100% 47%43% 45% 32% 100% 31% 29% 24% 16%

Example 4 Sample Preparation and RNA Extraction from Urine Samples

Urine sample (30 ml) was obtained from an individual with benignprostate hyperplasia was split into two 15 ml aliquots for extraction ofRNA from cellular components of urine sediment and soluble urinefractions.

The first aliquot of urine was processed for RNA extraction from thecells in the urine sediment using ZR Urine RNA Isolation Kit™ (ZYMOResearch Corporation). Briefly, cells were separated from urine by asyringe filter. The cells were retained on the syringe filter and thefiltrate was collected separately. The retained cells were lyseddirectly on the filter using 700 μl of RNA Extraction Buffer Plus™reagent (ZYMO Research Corporation) and the cell lysate was collected ina 1.5 ml tube. The cell lysate was mixed with an equal volume of ethanoland passed through Zima-Spin IC™ column. The column was washed with 300μl of RNA Wash Buffer. Total RNA was eluted from the column by applying25 μl of the supplied RNA Elution Buffer directly to the column membranefollowed by centrifugation.

The filtrate collected from the syringe filtration step described abovewas further concentrated using Amicon Ultra-15, nominal molecular weightlimit of 3 kDa (Millipore, Mass., USA) to a final filtrate volume of 500μl (soluble urine concentrate), representing approximately a 30-foldconcentration. The total nucleic acid was extracted from the solubleurine concentrate using NucliSENS® easyMAG® (bioMérieux, Inc., NC, USA)using manufacturer's protocol. Briefly, total nucleic acid binds toNucliSENS® magnetic silica particles. The magnetic silica particles wereseparated from the liquid portion using a magnetic field. The nucleicacid bound to silica particles were washed with the wash buffer providedby the manufacturer. The nucleic acid is finally released from the solidphase with the elution buffer. FIG. 1 (pathways 1 and 2) shows aschematic of the experimental design used to process the first urinealiquot (ultrafiltration step not shown).

The second urine aliquot (15 ml) was directly applied to an AmiconUltra-15, having a nominal molecular weight limit of 3 kDa (Millipore,Mass., USA). The urine sample was concentrated to 500 μl. The totalnucleic was extracted from the soluble urine concentrate usingNucliSENS® easyMAG® (bioMérieux, Inc., NC, USA) using manufacturer'sprotocol as discussed above. FIG. 1 (pathway 3) shows a schematic of theexperimental design used to process the second urine aliquot(ultrafiltration step not shown).

Example 5 cDNA Synthesis and RT-PCR

cDNA Synthesis and RT-PCR were performed in a one-step process using RNAUltraSense® one-step real-time (RT) PCR System (Invitrogen): First, RNAwas treated with DNase to eliminate DNA using RNA-free (Ambion). Amaster mix was prepared with following components for each reaction: RNAUltraSense Enzyme Mix 2.5 μl RNA UltraSense 5× Reaction Mix 10 μl,Taqman probe primer pair (10 μM concentration each) 1 μl, Fluorogenicprobe (10 μM) 1 μl, ROX Reference Dye 1 μl. Next, 3 μl of RNA templatein 31.5 μl of DEPC-treated water per reaction was added to a cleanmicrocentrifuge tube. The 34.5 μl of template was added to the 15.5 μlof Master mix for a total of 50 μl for each reaction. After gentlemixing, the reaction mixture was subjected to brief centrifugation, andwas placed in a preheated programmed thermal cycler. The instrument wasprogrammed to perform cDNA synthesis immediately followed by PCRamplification using the following cycling parameters: 50° C. for 15minute hold, 95° C. for 2 minute hold, 40-50 cycles of: 95° C. for 15seconds and 60° C. for 30 seconds. After cycling, the reaction was heldat 4° C. until further analysis.

Example 6 Estimation of Gene Expression Levels

Expression levels of four genes: heat shock 60 kDa protein 1 (HSPD1),inosine monophosphate dehydrogenase 2 (IMPDH2), PDZ and LIM domain 5(PDLIM5), and UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1); andfive reference genes: c-abl oncogene 1, receptor tyrosine kinase (ABL1),beta actin (ACTB), beta-2 microglobulin (B2M),glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta glucuronidase(GUSB) were evaluated using the RNA UltraSense® one-step RT-PCR System.Taqman® probes were used to monitor DNA synthesis. Fluorescent signalswere measured and plotted against the number of PCR cycles. The Ctvalue, the point at which the fluorescence crosses the baselinethreshold is measured for each gene. A lower Ct value indicates higherinitial concentration of template DNA and therefore initial RNA. The Ctvalues for four test genes and five reference genes were determinedusing the RNA isolated from cells present in urine, the urinesupernatant which is free of cells, and whole urine without furtherseparation of cells. The Ct values of different genes in various samplesare presented in Table 5 below.

TABLE 5 Ct values of the Genes in Different Samples Test Genes 5Reference Genes Kit Sample HSPD1 IMPDH2 PDLIM5 UAP1 ABL1 ACTB B2M GAPDHGUSB Zymo Urine 29.9 26.7 31.0 33.3 32.6 24.6 25.1 23.1 30.8 SedimentAmicon Urine 26.0 26.8 28.3 31.9 30.7 23.9 26.5 22.1 28.8 SupernatantSoluble 26.6 24.8 27.1 30.9 29.8 21.4 23.0 20.6 27.3 Urine Concentratefrom Whole Urine (3K) Soluble 29.2 28.2 30.5 32.8 33.2 24.6 27.0 24.430.3 Urine Concentrate from Whole Urine (10K)

The results in Table 5 demonstrate that the amount of RNA in whole urineis generally higher than that obtained from the cells present in urine.Additionally, the urine supernatant contains more transcript than thecells in urine sediment for the majority of the genes tested.Furthermore, the expression pattern of the five reference genes variedamong the cells in urine sediment, urine supernatant (after separationof cells) and soluble urine concentrate (without separation of cells).As seen in Table 5, concentration of whole urine to form a soluble urineconcentrate consistently yielded higher amounts of RNA when a membranewith smaller pore size was used (cf. MW=3K versus MW=10K cutoff).

Example 7 Assessment of Diagnostic RNA for Prostate Cancer in UrineFractions

Urine Collection and Processing:

Urine samples were collected from male individuals diagnosed as havingprostate cancer prior to a prostate biopsy. Urine samples were collectedfrom the study participants using standard urine collection cups. Eachurine sample was split in order to obtain fractions of whole urine,urine sediment, and urine supernatant from each study individual. Urinesupernatant was produced by filtering 15 ml of whole urine using anAmicon Ultra-15, nominal molecular weight limit of 3 kDa (Millipore,Mass., USA). Whole urine and urine sediment was processed as describedabove. RNA from all urine fractions was isolated using the ZR Urine RNAIsolation Kit™ (ZYMO Research Corporation) and subsequently processed,concentrated, and processed as described above. DNA was removed from theresulting nucleic acids using the DNA-free kit (Ambion, Cat. # AM1906)according to the manufacturer's protocol.

RT-PCR Amplification of Urinary RNA:

RNA from each urine fraction was quantified for the amount ofprostate-specific antigen (PSA), HSPD1, UAP1, IMPDH2, and PDLIM5, andGAPDH as a control gene, by RT-PCR. The specific primers and probes areprovided in Table 6. IMPDH2 and PDLIM5 were assessed using a TaqMan®Gene Expression Assay according to the manufacturer's protocols (AppliedBiosystems; Cat. Nos. Hs01021353_m1 and Hs00935062_m1, respectively).qRT-PCR assays were run on an ABI 7900HT instrument with the followingthermocycling conditions: Step 1: 50° C., 20 min.; Step 2: 94° C., 2min.; Step 3: 94° C., 15 sec.; Step 4: 58-60° C., 30 sec.; cycle steps3-4 forty five times.

TABLE 6 RT-PCR Primers/Probes For Prostate Cancer-specific Genes SEQ IDPrimer/Probe Sequence NO: GAPDH-F 5′-GAA GGT GAA GGT CGG AGT C-3′ 1GAPDH-R 5′-GAA GAT GGT GAT GGG ATT TC-3′ 2 GAPDH-Probe5′-FAM-CAA GCT TCC CGT TCT CAG CC-BHQ-3′ 3 HSPD1-F5′-AAC CTG TGA CCA CCC CTG AA-3′ 4 HSPD1-R5′-TCT TTG TCT CCG TTT GCA GAA A-3′ 5 HSPD1-Probe5′-VIC-ATT GCA CAG GTT GCT AC-BHQ-3′ 6 UAP1-F5′-TTG CAT TCA GAA AGG AGC AGA CT-3′ 7 UAP1-R5′-CAA CTG GTT CTG TAG GGT TCG TTT-3′ 8 UAP1-Probe5′-VIC-TGG AGC AAA GGT GGT AGA-BHQ-3′ 9

Quantitation of Urinary RNA:

The amount of HSPD1, IMPDH2, PDLIM5, and UAP1 were normalized to eitherthe concentration of GAPDH RNA or PSA RNA. A seven-point standard curvefor PSA was generated using PSA concentrations of 0.0122-50.0 ng/μl.Normalized diagnostic gene scores were calculated according to thefollowing formula:Normalized Diagnostic Gene Score=Ln(Diagnostic Gene RNA/Control GeneRNA)wherein the Diagnostic Genes are HSPD1, IMPDH2, PDLIM5, and UAP1,individually, and the Control Gene is either GAPDH or PSA. A combined4-gene score was calculated by summing the four Normalized DiagnosticGene scores for each of the GAPDH and the PSA normalizations.

Results:

The results of this study are illustrated in FIG. 2. Levels of PSAtranscript were found to be highest in the soluble urine fraction(supernatant). Additionally, the amount of individual diagnostic genetranscripts and the diagnostic 4-gene score was also found to be highestin the soluble urine fraction indicating that this fraction provides thegreatest diagnostic sensitivity and represents a significant improvementover the transcript levels detected in the urine sediment.

Example 8 Assessment of Diagnostic RNA for Prostate Cancer in UrineFractions

Urine Collection and Processing:

Blood and urine samples were collected from male individuals diagnosedas having prostate cancer prior to a prostate biopsy, diagnosed ashaving benign prostate hyperplasia (BPH), or identified as not havingeither prostate cancer or BPH. Prostate cancer patients were maleshaving a positive diagnosis, ages 50-75, and within 30 days of ascheduled prostate biopsy. Exclusion criteria from the patientpopulation included a history of prior prostate cancer treatment or aprevious diagnosis of any other cancer within the last five years(excluding non-melanoma skin cancer). The control populations weremales, 50-75 years of age, and having a PSA result within the six monthsprior to sample collection of ≥1.0 ng/mL. Exclusion criteria from thecontrol population included any prior diagnosis and/or treatment forprostate cancer or any other cancer, excluding non-melanoma skin cancer.Control subjects had to otherwise feel healthy and have no evidence offever. Urine samples from all individuals was collected and processed asdescribed in Example 7.

Four-Gene Score:

The diagnostic genes HSPD1, UAP1, IMPDH2, and PDLIM5, along with PSAwere amplified and quantified from whole urine and urine supernatant(soluble urine fraction) of prostate cancer (PCa) and BPH patientsamples, and the 4-gene score was calculated. All methods andcalculations were performed as described in Example 7. The 4-gene scoreresults from individual samples are provided in FIG. 3.

The diagnostic potential of various cut-off scores was investigated.Applying a diagnostic cut-off score of 2.6 to the data set in FIG. 3resulted in the correct identification of 63% of PCa patients, whileonly 25% of BPH patients showed an elevated 4-gene score in the urinesupernatant. A cut-off score of 5.0 resulted in the correctidentification of 33% of PCa patients while only 15% of BPH patients had4-gene scores greater than this level. When these cutoff levels wereapplied to the 4-gene scores determined in whole urine, the 2.6 levelcorrectly identified 61% of PCa patients while 52% of BPH patientsshowed an elevated score. The 5.0 cutoff level resulted in 43% of PCasamples being correctly identified, whereas only 13% of BPH samples wereelevated.

PSA and PCA3 Levels in Whole Urine and Urine Fractions:

The relative amounts of other prostate cancer markers, PSA and PCA3,were investigated in the three urine fractions. In each fraction, theamount of PSA transcript was quantified by RT-PCR, as above, andnormalized to GAPDH, as above. For convenience, the PSA level wasmultiplied by a factor of ten before GAPDH normalization. As shown inFIG. 4, normalized PSA levels (nPSA) were highest in whole urine andurine supernatant, which were significantly greater than nPSA levels inthe urine sediment.

The Ct value for the PCA3 transcript, measured during the RT-PCRreaction was used as a relative index of the PCA3 transcriptconcentration present in the sample. The Ct value, in a quantitative PCRsuch as that used here, is the amplification cycle at which the detectedfluorescence crosses a pre-determined threshold value. Thus, lower Ctvalues are indicative of higher starting concentrations of targetnucleic acid because fewer cycles of amplification are required to reachthe threshold. For convenience, the PCA3 data presented in FIG. 4 is the45-Ct_(observed) in the PCA3 assay. This transformation illustratesgraphically increasing values proportional to increasing starting PCA3concentrations (FIG. 4). As was the case for nPSA, PCA3 RNA levels werehighest in whole urine and urine supernatant, each of which wassignificantly higher than the levels measured in the urine sediment.

Validation of the Soluble Urine Fraction for PCa Diagnosis:

In order to determine the relative diagnostic capacity of the solubleversus insoluble urine fractions, and whole urine, for PCa diagnosis,the 4-gene score for each patient was determined using PSA normalizationaccording to the formulas provided in Example 7. The median 4-gene scorewas determined for each fraction and each disease state (PCa and BPH).For each urinary fraction, the 4-gene score for BPH was subtracted fromthe 4-gene score for PCa. The median differences are illustratedgraphically in FIG. 5. As shown for whole urine, the median 4-gene scorefor PCa and BPH were approximately the same, indicating that whole urineis generally not suitable for differential PCa diagnosis using thepresent methods. Differences in the median 4-gene score using urinesediment samples indicate that BPH generally results in higher scoresthan PCa which has the potential to result in a high level of falsepositive PCa diagnoses. In contrast, the median 4-gene score determinedin urine supernatant was greater for the PCa samples than for the BPHsamples indicating that the urine supernatant has a greater capacity tobe used for differential diagnosis of prostate disease.

Example 9 Clinical Study of Six-Gene Assay for Detecting Prostate Cancer

Urine was collected from 125 male subjects. The group included 50prostate cancer (PCa) patients with urine collected before and afterprostatectomy (cohort 1), 25 healthy men with serum PSA values below 1.0ng/ml deemed unlikely to harbor prostate cancer (cohort 2), and 50 menwith serum PSA values greater than 2.5 ng/ml who were scheduled forprostate biopsy (cohort 3). A subgroup of 25 men from cohort 3 had urinesamples collected before and after digital rectal exam (DRE) (cohorts 4and 5 respectively) but before biopsy. Serum from each patient was alsocollected and analyzed for PSA.

Urine from each subject was aliquoted into 30 mL fractions, with onefraction each for assaying supernatant, sediment, and whole urine, aswell as aliquots sent to an outside laboratory to test for PCA-3.Extraction of RNA for the supernatant, sediment, and whole urinefractions was completed immediately, as described in Example 7. Eachpatient fraction was analyzed for the amount of four target genes andtwo reference genes using RT-PCR, as described in Example 5. The targetgenes were PDLIM5, UAP1, HSPD1, IMPDH2, and the reference genes were PSAand GAPDH. Assay results were blinded and data was correlated with theclinical findings after all assays were completed.

Example 10 Data Analysis of Urine from Clinical Study

Data from a subset of urine supernatant samples collected before DRE, asdescribed in Example 9, were analyzed for the ability to detect prostatecancer. The supernatant samples used in the study had PSA mRNA >0.001 ngand a combined 4-target gene plus PSA mRNA >0.1 ng. The study size of 40patients (15 PCa and 25 benign) was powered to detect an area under thereceiver operating characteristic curve (AUROC) of 0.70 with ≥58%accuracy. The resulting AUROC was 0.544 (p-value=0.685). The AUROCvalues for the entire 4-gene panel as well as each of the individualgenes in urine supernatant, urine sediment, and whole urine aresummarized in Table 7 below.

TABLE 7 Pre-DRE AUROC Values to Distinguish PCa from Non-PCa GeneTarget/Ref. AUC (Sensitivity/Specificity) Gene Supernatant SedimentWhole 4-gene/PSA 0.544 (33%/68%) 0.527 (50%/55%) 0.516 (36%/72%)HSPD1/PSA 0.564 (60%/60%) 0.530 (33%/77%) 0.505 (43%/76%) IMPDH2/PSA0.573 (47%/60%) 0.538 (58%/50%) 0.552 (50%/60%) PDLIM5/PSA 0.508(40%/72%) 0.610 (58%/68%) 0.513 (43%/56%) UAP1/PSA 0.500 (53%/52%) 0.506(50%/55%) 0.510 (50%/60%) 4-gene/ 0.552 (60%/64%) 0.558 (55%/68%) 0.605(86%/52%) GAPDH HSPD1/ 0.573 (67%/56%) 0.550 (55%/59%) 0.594 (57%/63%)GAPDH IMPDH2/ 0.609 (67%/56%) 0.574 (45%/67%) 0.618 (86%/57%) GAPDHPDLIM5/ 0.523 (47%/64%) 0.608 (45%/58%) 0.575 (64%/46%) GAPDH UAP1/GAPDH0.560 (60%/60%) 0.525 (55%/59%) 0.568 (71%/50%)

Urine from post-DRE samples were also analyzed, with sample numbers of23, 20, and 19 for supernatant, sediment, and whole urine, respectively.Most AUROC values were not significantly above 0.50 except forIMPDH2/PSA in urine sediment, which had an AUROC value of 0.766. Table 8below summarizes AUROC data for urine collected after DRE.

TABLE 7 Post-DRE AUROC Values to Distinguish PCa from Non-PCa GeneTarget/Ref. AUC (Sensitivity/Specificity) Gene Supernatant SedimentWhole 4-gene/PSA 0.571 (44%/57%) 0.688 (75%/58%) 0.583 (57%/42%)HSPD1/PSA 0.595 (33%/71%) 0.625 (63%/58%) 0.607 (43%/50%) IMPDH2/ 0.540(56%/50%) 0.766 (75%/67%) 0.530 (71%/42%) PSA PDLIM5/ 0.587 (33%/43%)0.609 (50%/67%) 0.607 (43%/58%) PSA UAP1/PSA 0.595 (56%/36%) 0.641(63%/58%) 0.595 (43%/42%) 4-gene/ 0.650 (67%/71%) 0.563 (50%/75%) 0.583(57%/50%) GAPDH HSPD1/ 0.670 (78%/71%) 0.583 (50%/75%) 0.589 (43%/83%)GAPDH IMPDH2/ 0.686 (67%/79%) 0.615 (50%/75%)  0.667 (43%/100%) GAPDHPDLIM5/ 0.664 (78%/64%) 0.583 (50%/75%)  0.613 (29%/100%) GAPDH UAP1/0.664 (78%/57%) 0.563 (50%/75%) 0.637 (57%/75%) GAPDH

In the same sample subset, the PCA3 assay yielded an AUROC of 0.711.Using a PCA3 cutoff value of 35, the specificity and sensitivity forPCA3 detection were 60% and 75%, respectively.

Example 11 Data Analysis of Urine Supernatant and Urine Sediment fromClinical Study

Data from the study described in Example 9 was re-analyzed, focusing onthe quality control parameters for RNA concentration. The analysisdescribed in Example 10 included samples with PSA mRNA >0.001 ng and acombined 4-target gene plus PSA mRNA >0.1 ng. For all subjects whoprovided one urine sample, an average of 28% of the sediments wereexcluded, as were 15% of the supernatants and 17% of the whole urines.However, a review of the post-prostatectomy urine samples showed that 28of the 49 post prostatectomy urines met the criteria of a quality samplehaving an adequate PSA RNA concentration, when in fact the PSA RNAvalues for these urines should have been zero. The PSA copy number wasless than the detectable value of 3000 copies per mL in all of thepost-prostatectomy sample types, confirming the absence of detectableRNA. Thus, the criteria for defining a quality sample for urinesupernatant was set too low, allowing background noise to be accepted asan actual value.

In the post-prostatectomy urine supernatant samples, the highest valueobserved for PSA mRNA was 0.068. Thus a new criteria of 0.07 ng wasselected to redefine these urine samples as not detectable for PSA RNA.The criteria of 0.001 ng for urine sediment was maintained for judgingthe acceptability of urine sediments for assay, as all of thepost-prostatectomy urine sediments had undetectable PSA RNA.

Using these sample selection criteria for urine supernatant and sedimentRNA, a subset of 46 supernatant and sediment samples were selected. An Sscore (sum of target gene expression normalized by the two referencegenes) for this subset of samples was calculated from a linear SVMequation using the six gene expression inputs for each urine supernatantand sediment. The linear SVM is a form of the normalization equationthat was published for the tissue gene test and which was used in theanalysis of the data as described in Example 10. The S score wascalculated for the supernatant and for its matching sediment for allurines with a supernatant PSA RNA greater than 0.07 ng. A positiveclassification of cancer was defined as either the supernatant orsediment being positive. A negative classification of cancer requiredboth the supernatant and sediment to have a negative S score.

The classification achieved with the S score gave a sensitivity of 80%and specificity of 83%. An ROC curve was constructed to demonstrate theaverage performance over the range of S cut-off values and had an areaunder the curve of 0.813.

Matching data for PCA3 detection at a cutoff value of 35 gave asensitivity of 55% and a specificity of 88%. In the entire study groupof 125 subjects, in which 43 subjects were excluded due to low copynumber for either PCA3 or PSA, the PCA3 detection sensitivity of theremaining subgroup was 58% with a specificity of 74%.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Additionally, the terms andexpressions employed herein have been used as terms of description andnot of limitation, and there is no intention in the use of such termsand expressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.

Thus, it should be understood that although the invention has beenspecifically disclosed by preferred embodiments and optional features,modification, improvement and variation of the inventions embodiedtherein herein disclosed may be resorted to by those skilled in the art,and that such modifications, improvements and variations are consideredto be within the scope of this invention. The materials, methods, andexamples provided here are representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

All publications, patent applications, patents, and other referencesmentioned herein are expressly incorporated by reference in theirentirety, to the same extent as if each were incorporated by referenceindividually. In case of conflict, the present specification, includingdefinitions, will control.

What is claimed is:
 1. A detection method comprising: (a) separatingurine sediment from a soluble urine fraction of a urine sample obtainedfrom an individual suspected of having prostate cancer; (b)concentrating the soluble urine fraction by ultrafiltration to produce asoluble urine concentrate, wherein the volume of soluble urineconcentrate is reduced at least 50% from the original urine volume; (c)detecting the amount of RNA from each of heat shock 60 kDa protein 1(HSPD1), inosine monophosphate dehydrogenase 2 (IMPDH2), PDZ and LIMdomain 5 (PDLIM5), and UDP-N-acetylglucosamine pyrophosphorylase 1(UAP1) in the concentrated soluble urine fraction.
 2. The method ofclaim 1, wherein the ultrafiltration comprises filtration using a filterhaving a nominal molecular weight limit of not more than about 50,000daltons.
 3. The method of claim 1, wherein the ultrafiltration comprisesfiltration using a filter having an nominal molecular weight limit ofnot more than about 3,000 daltons.
 4. The method of claim 1, wherein thesoluble urine fraction produced by the ultrafiltration is cell-free. 5.The method of claim 1, further comprising isolating the RNA from thesoluble urine concentrate by solid phase extraction.
 6. The method ofclaim 1, wherein the amount of each said RNA present in the solubleurine fraction is determined by reverse-transcriptase PCR (RT-PCR). 7.The method of claim 6, wherein the determination by RT-PCR is performedin real-time.
 8. The method of claim 6, wherein the Ct value is used todetermine the amount of each said RNA.
 9. The method of claim 1, whereinthe amount of RNA is normalized to the amount of a control gene RNA. 10.The method of claim 9, wherein the control gene RNA encodes a geneselected from the group consisting of prostate-specific antigen (PSA),c-abl oncogene 1, receptor tyrosine kinase (ABL1), beta actin (ACTB),beta-2 microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase(GAPDH), and beta glucuronidase (GUSB).
 11. The method of claim 1,further comprising detecting the amount of RNA encoding prostate cancerantigen-3 (PCA3) in the concentrated soluble urine fraction.